The long term goal of this proposal is directed towards understanding how members of the CIC CI-channel family, in conjunction with CFTR, function to provide CI- transport across airway epithelia. These alternative channels may provide other routes for CI- transport when CFTR is defective. Two CIC CI- channels have been identified in adult and fetal human lung, CIC-2G (also named CIC-2 alpha) and CIC-3. CIC- 2G has been cloned from rabbit stomach and human lung by this laboratory, and show PKA and CaMKII regulation and regulation by extracytoplasmic pH. Both CIC-2G and CIC-3 channels may be involved in regulated CI- transport in the human lung. This proposal is to determine the physiological, functional, structural, and regulatory properties of CIC CI- channels in the human lung. The specific aims are to: 1) Identify the CI- channel forms, determine the levels and location of CIC-2G and CIC-3 in the human lung. This aim is built upon the finding of CIC-2G and CIC-3 in the human lung, and quantitative RT-PCR results which demonstrate the presence of these channels in the adult as well as in the fetal human lung. Quantitative RT-PCR, peptide antibodies and in situ hybridization will be used to quantitate the distribution of CIC channels in the human and rabbit lung; 2) Determine the functional significance of consensus phosphorylation sites in the human lung CIC-2G CI-channel. This aim is build upon the finding of additional consensus PKA and CaMKII sites in human CIC-2G, activation of human CIC-2G CI-channel function by these protein kinases, and lack of activation in mutants lacking these sites; 3) Define the functional significance of CIC-2G CI- channels in transfected epithelial cells will be compared and contrasted with the properties of endogenous channels from human airway epithelial cells which contain CIC channels using patch clamp. This includes effects of protein kinases (PKA and CaMKII) and low extracytoplasmic pH. Mild chemical procedures for channel activation using amidation catalysed by water soluble carbodiimides will be investigated as a novel approach to development of pharmaceuticals for treatment of cystic fibrosis patients.